Human Chronic Myelogenous Leukemia Research Articles

C-Myc Is Required for Acute but Not for Chronic Hematopoietic Malignances in Pten-Null Mice.

Abstract 1632Poster Board I-658c-Myc, the product of an oncogene, is a common target of most leukemic oncoproteins. Deregulation of c-Myc is commonly found in human leukemic blasts. Transgenic over-expression of c-Myc induces myeloid, erythroid, and lymphocytic leukemia in mice, however whether c-Myc is absolutely required for leukemogenesis has not yet been addressed.Pten, a tumor-suppressing phosphatase, inhibits cell proliferation and promotes apoptotic cell death through repression of PI3K-Akt signaling. Inactivating mutations of Pten and deregulation of PI3K/Akt signaling are both involved in the development of both chronic and acute hematopoietic malignances. Mice with Pten deletions in hematopoietic stem cells (HSCs) develop myeloproliferative disorders (MPD) followed by acute T lymphocytic or myeloid leukemia, reminiscent of disease progression in human chronic myelogenous leukemia. Mice with Pten deletions in lymphocytes develop lymphadenopathy (due to a chronic polyclonal lymphoproliferative disorder) as well as CD4+ T lymphocytic lymphoma. The appearance of these diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously.To study whether c-Myc is required for the development of these hematopoietic disorders in Pten-mutant mice, we generated inducible Pten and c-Myc double-knockout mice (Pten-/-c-Myc-/-). By comparing the hematopoietic phenotypes of the Pten-/-c-Myc-/- mice with those of Pten-mutant (Pten-/-) mice, we found that both sets of mice developed MPD and lymphadenopathy, however none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPD which developed in Pten-/- mice, which is dominated by granulocytes, megakaryocytes predominate in the MPD of Pten-/-c-Myc-/- mice.We have concluded that c-Myc is required for the development of both T lymphocytic lymphoma and the acute leukemic transformation of Pten-/- MPD, but is not essential for the development of chronic myeloid or lymphoid proliferative disorders. Our study suggests that deregulation of PI3K/Akt signaling in Pten-mutant hematopoietic cells protects these cells from apoptotic cell death, which results in chronic proliferative disorders, while the deregulation of c-Myc resulting from additional mutations promotes hematopoietic cell proliferation and blockage of maturation, and is absolutely required for the development of acute hematopoietic malignances. DisclosuresNo relevant conflicts of interest to declare.

A Common but Overlooked Mechanism of BCR-ABL1 Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

Abstract 2179Poster Board II-156Point mutations in the BCR-ABL1 protein are the most commonly reported mechanism of resistance to imatinib mesylate (Gleevec or STI-571) treatment in patients with chronic myelogenous leukemia (CML). We have reported that some patients with imatinib-resistant CML express an alternatively spliced BCR-ABL1 mRNA with a 35-bp insertion (BCR-ABL135INS), but the prevalence and effect of this mutation have not been well delineated. We sought to improve understanding of this mutation in order to provide insight into mechanisms of resistance to tyrosine kinase inhibitors. Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in a group of 168 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot in patient samples. The effects of various kinase inhibitors on human K562 CML cells transfected with BCR-ABL135INS cDNA were tested by measuring cell survival, caspase-3 activation, and the BIM activation pathway. In our analysis of BCR-ABL1 genotypes in 168 imatinib-resistant chronic-phase CML patients, (56%) of patients expressed various levels of BCR-ABL135INS whereas only (25%) had detectable point mutations in BCR-ABL1. Expression of this truncated BCR-ABL1 fusion protein was also confirmed by immunoprecipitation at levels proportional to those predicted by mRNA quantification. In vitro, BCR-ABL135INS expression conferred resistance to the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib in a concentration-dependent fashion, but had no effect on the protein-synthesis inhibitor homoharringtonine (HHT or omacetaxine) or the aurora kinase inhibitor MK-0457 (VX-680). The combination of imatinib with nilotinib or HHT was synergistic in overcoming BCR-ABL135INS-induced resistance. Moreover, the resistance to imatinib induced by BCR-ABL135INS was associated with loss of BIM expression. This loss of BIM was overcome by pretreatment with HHT. In conclusion, these findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS found in imatinib-resistant CML patients. Optimal patient management in the future will likely require periodic testing for the expression of BCR-ABL-135INS mRNA during therapy. This may help in predicting various levels of resistance or the potential for eliminating the disease by adjusting the treatment strategy. Disclosures:No relevant conflicts of interest to declare.

The Effect of siRNA Down-Regulating XIAP On the Apoptosis and Chemo-Sensitivity of K562 Cells in Vitro.

Abstract 5053 IntroductionFailure for chemotherapy in leukemia patients is often due to innate or acquired multi-drug resistance of leukemia cells. The X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of apoptosis and is also involved in drug resistance. The relationship between XIAP and drug resistance is not fully clarified. Here, we manipulated the expression of XIAP in K562 human chronic myelogenous leukemia cells and explore the changes in cell apoptosis and sensitivity to cytarabin. MethodsSmall interfering RNA (siRNA) targeting XIAP was created, K562 cells were either transfected with XIAP siRNA or control siRNA by Nucleofector transfection. The siRNAs were labelled with Cy3. Real-time RT-PCR and Western blot were used to determine the expression of XIAP in K562 cells. Flow cytometry was used to detect apoptotic cells and 50% inhibiting concentration (IC50) of cytarabin was also determined. ResultsThe transfection rate was 87% in general. The transfection was also confirmed by fluorescence microscopy. After 48h, the expression levels of XIAP mRNA and protein were much lower in XIAP siRNA-transfected cells than in control siRNA-transfected cells (mRNA, 0.37±0.10 vs. 1.41±0.13, P<0.05 ; protein, 0.39±0.03 vs. 0.99±0.08, P<0.05). There was no significant difference in the fractions of apoptotic cells between these two groups at 48h after transfection (37.04%±1.77% vs. 47.43%±1.13%, P>0.05). Cytarabin was added in different concentrations right after Nucleotransfection. After 48h, the IC50 of Cytarabin in the XIAP siRNA-transfected cells was 2.28-fold lower than that in the control siRNA-transfected cells (82.60±21.56μg/ml vs. 188.67±44.48μg/ml, P<0.05). The percentage of apoptotic cells was much higher in the XIAP knockdown cells than in the control cells (47.43%±1.13% vs. 37.04%±1.77% P<0.05). ConclusionsXIAP siRNA can specifically down-regulate the expressions of XIAP mRNA and protein, promote the drug-sensitivity to cytarabin and increase the cell apoptosis in K562 cells. This might be a novel choice for leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Physiologic Hypoxia Protects Chronic Myelogenous Leukemia Progenitors From Elimination by Imatinib Mesylate.

Abstract 2181Poster Board II-158The use of Abl tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has seen a major advance in the control of chronic myelogenous leukemia (CML). However, since TKIs do not seem to eliminate CML stem cells, the use of TKIs may not represent a curative approach. Recent reports have shown that the bone marrow (BM) microenvironment, which facilitates the proliferation and renewal of resident hematopoietic stem cells (HSC), is hypoxic. The average BM O2 tension has been measured at 6-7% O2 in humans (physiologic hypoxia), including in patients with leukemia (Fiegl et al. Blood, 2009), while levels in the HSC niche are estimated to be even lower (<1% O2). In light of the above, it is likely that CML stem cells also reside in the HSC niche, and are similarly adapted for survival and self-renewal in this environment. Furthermore, although hypoxia is known to be associated with both radio- and chemo-resistance in solid tumors, its role in leukemia has has not been thoroughly investigated, particularly in the context of resistance to targeted therapies. Accordingly, we hypothesized that hypoxia protects CML progenitor cells from elimination by IM, a phenomenon which may contribute to disease persistence. To test the above, we examined if hypoxia modified the response of CML progenitor cells toward IM. Primary chronic phase (CP) CML samples “from either BM or peripheral blood (PB)” were obtained at the time of diagnosis from seven individuals with clinically-defined IM-sensitive (IM-S) or IM-resistant (IM-R) disease. Cells were then incubated with IM (0, 0.25, 1 or 5μM) under hypoxic (0.5% O2) or normoxic (21% O2) conditions for 96 hours. The treated cells were then harvested and plated in methylcellulose for 14 days (under 21% O2), after which the number of colony-forming cells (CFCs) were counted by two independent observers. In the absence of IM, four of the seven samples had a significant increase in CFCs (1.5-2.4 fold) when cultured in 0.5% O2 vs 21% O2, suggesting that primary CML CFCs may be better maintained in hypoxia. Furthermore, when treated with IM, six out of seven samples cultured under 0.5% O2 demonstrated dramatic increases in CFCs compared to those treated under 21% O2: a 2.3 to 9.0-fold increase at 1μM, and a 4.4 to 35.0-fold increase at 5μM IM. We also found that the protective effect of hypoxia was independent of the original source of the CFCs (BM or PB). In addition, we found that progenitors from both IM-S and IM-R patients were protected from IM under 0.5% O2, suggesting that hypoxia-induced protection is a general feature of CML progenitors. In order to establish a model for further study, we also tested if hypoxia elicits similar responses in four human CML cell lines (K562, AR230, LAMA84, and BV173). In contrast to primary CML cells, we found that hypoxia actually impaired the ability of all four cell lines to form colonies, and also did not confer protection from IM. These results show that CML cell lines have adapted to conditions of normoxia, and are thus inappropriate models to study the hypoxic response in CML. Additional data will be presented describing the mechanisms that may underlie the protective effect of hypoxia, as well as compounds that can counteract such effects. Ongoing experiments using the LT-CIC assay will also determine the response of primitive CML progenitors to growth under conditions of physiologic hypoxia vs normoxia. In conclusion, our results show that physiologic hypoxia protects CML progenitors from IM, and suggest that blocking of hypoxia-induced survival pathway(s) in CML progenitor cells may facilitate the elimination of residual CML progenitors. Disclosures:No relevant conflicts of interest to declare.

Gene Therapy of Chronic Myelogenous Leukemia Using Pseudotyped Recombinant Adeno-Associated Viral Vectors.

Abstract 4506 IntroductionGene transfer into malignant leukemia cells can be of relevance to overcome conventional therapy-resistance. Either suicide or immune stimulating gene therapy vectors may be a tool for second line treatment of imatinib-resistant chronic myelogenous leukemia (CML). Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency. MethodsPseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) were screened on a panel of human CML cell lines and primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations. ResultsOn CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient (Fig. 1). For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells were observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p=0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p<0.001 vs rAAV2/6; Fig. 2). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors. Furthermore, donor-dependent differences in gene transfer efficiency were observed. Of note, data from experiments on CML cell lines seems to provide limited information about the transduction efficiency of rAAV vectors on primary CML cells. ConclusionsOverall, pseudotyped rAAV and scAAV vectors offer efficient clinically relevant gene transfer into human CML cells with a potential for future clinical application. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hemin Counteracts the Repression of Bcl-2 and Nrf2 Genes and the Cell Killing Induced by Imatinib in Human Bcr-Abl(+) CML Cells

Imatinib is a targeted selective inhibitor of chimaeric Bcr-Abl tyrosine kinase developed for effective therapy of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) patients. Unfortunately, evidence now exists to indicate that a portion of such patients treated with imatinib acquire resistance and subsequently relapse. To understand the heterogeneous basis of imatinib resistance, we have investigated the possible mechanism(s) via which hemin, a key regulator of hematopoiesis that is converted to heme intracellularly, renders CML cells less susceptible to imatinib. Hemin at 30-90 aM protected a substantial proportion (>40%) of human Bcr-Abl(+) CML cells (K-562 and KU-812) from imatinib-induced cell killing by increasing the imatinib IC50 value, reducing DNA damage, and promoting erythroid differentiation. RT-PCR assessment of RNA transcripts encoded by human GAPDH, Ggamma-globin, Bcr-Abl, HO-2, Hpr-6, CEBPa, Bcl-2a, Bcl-2b, and Nrf2 genes revealed that hemin selectively counteracted the repression of antiapoptotic Bcl-2a, Bcl-2b, and Nrf2 genes in imatinib-treated cells. These genes are markedly repressed by imatinib alone in human K-562 CML cells. Hemin, however, had no detectable effect on the expression of the Bcr-Abl gene. Moreover, inhibition of de novo heme biosynthesis by succinyl-acetone enhanced the killing effect of imatinib. These data clearly indicate that: (a) cellular heme resulted from de novo biosynthesis and hemin uptake alters the developmental stage of human Bcr-Abl(+) CML cells and their susceptibility to imatinib; (b) cellular heme counteracts the ability of imatinib to repress Bcl-2 and Nrf2 gene expression; and (c) inhibitors of de novo biosynthesis can be developed and combined with imatinib to enhance its antileukemic activity.

Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: Synthesis and biological evaluation of antivascular activity

The α-methyl chalcone SD400 is a potent inhibitor of tubulin assembly and possesses potent anticancer activity. Various chalcone analogues were synthesized and evaluated for their cell growth inhibitory properties against the K562 human chronic myelogenous leukemia cell line (SD400, IC 50 0.21 nM; combretastatin A4 CA4, IC 50 2.0 nM). Cell cycle analysis by flow cytometry indicated that these agents are antimitotic (SD400, 83% of the cells are in G 2/M phase; CA4 90%). They inhibit tubulin assembly at low concentration (SD400, IC 50 0.46 μM; CA4, 0.10 μM) and compete with [ 3H]colchicine for binding to tubulin (8% [ 3H]colchicine remained bound to tubulin after competition with SD400 or CA4). Upon treatment with SD400, remarkable cell shape changes were elicited in HUVEC cells, consistent with vasculature damaging activity.

Effect of Curcumin on Nuclear Factor κB Signaling Pathways in Human Chronic Myelogenous K562 Leukemia Cells

Curcumin, a natural product isolated from the plant Curcuma longa, has a diverse range of molecular targets that influence numerous biochemical and molecular cascades. Curcumin has been shown to inhibit nuclear factor kappaB (NF-kappaB) activation at several steps in the NF-kappaB signaling pathways and thereby controls numerous NF-kappaB-regulated genes involved in various diseases. In the present study, we investigated the effect of curcumin pretreatment on 84 tumor necrosis factor-alpha (TNF-alpha)-activated genes of NF-kappaB pathways in K562 cells, using a real-time PCR array. Our results show that transcription of 29 NF-kappaB-related mRNAs was significantly downregulated (CARD4, CCL2, CD40, CSF2, F2R, ICAM1, IKBKB, IKBKE, IL1A, IL1B, IL6, IL8, IRAK2, MALT1, MAP3K1, MYD88, NFKB1, NFKB2, NFKBIA, PPM1A, RAF1, RELB, STAT1, TLR3, TNF, TNFalphaIP3, TNFSF10, and TICAM1), whereas 10 mRNAs were induced (AGT, CASP1, CSF3, FOS, IFNG, IL10, TICAM2, TLR2, TLR9, and TNFRSF7). Western blot analysis of CD40, NFKB1 (p50), RELB, NFKBIA (IkappaBalpha), and IL10 as well as an IL8 secretion assay confirmed our results. Taken together, we show that curcumin regulates an impressive number of NF-kappaB genes within the different NF-kappaB signaling pathways.

Docetaxel enhances the cytotoxic effects of imatinib on Philadelphia positive human chronic myeloid leukemia cells

Chronic myelogenous leukemia (CML) results from a translocation between chromosomes 9 and 22 which generates BCR/ABL fusion protein and characterized by uncontrolled proliferation of immature white blood cells. Imatinib, a molecularly targeting anticancer agent, is used widely for the treatment of CML and showed significant activity in chronic and accelerated phases but much less in blast crisis phase. The resistance to imatinib especially in blast crisis phase is recognized as a major problem in the treatment of CML patients. Docetaxel is shown to arrest cells in G2/M phase of the cell cycle which makes cells more sensitive to chemo- and radiotherapy. In this study, we aimed to increase chemosensitivity of human K562 CML cells to imatinib in combination with docetaxel. Taken together, our results showed that the combination of imatinib and docetaxel decreased cellular proliferation and increased apoptosis in human K562 chronic myeloid leukemia cells as compared to any agent alone. Imatinib and docetaxel induced apoptosis through caspase-3 enzyme activity and mitochondrial membrane potential.

Phytochemical, Antibacterial, Antiproliferative, and Antioxidant Potentials and DNA Damage-Protecting Activity ofAcacia salicinaExtracts

The extract enriched in total oligomer flavonoids (TOF), and the aqueous, methanol, and ethyl acetate extracts of Acacia salicina were investigated for their polyphenolic compound content, antioxidative activity in the nitro blue tetrazolium/riboflavin assay system, antibacterial activity against Gram-positive and Gram-negative bacterial reference strains, antigenotoxic activity tested with the Ames assay, and cytotoxic activity against the K562 human chronic myelogenous leukemia cell line and L1210 leukemia murine cells. TOF extract was effective at inhibiting nitro blue tetrazolium reduction by superoxide radical in a nonenzymatic O(2)(*-)-generating system. Significant activity against bacterial reference strains Staphylococcus aureus, Enterococcus faecalis, Salmonella enteritidis, and Salmonella typhimurium was shown with all the tested extracts. These extracts significantly decreased the genotoxicity induced by sodium azide and 4-nitro-o-phenylenediamine. A pronounced cytotoxic effect on both leukemia cell lines was detected in TOF, methanolic and ethyl acetate extracts. The antioxidant, antimicrobial, antigenotoxic, and cytotoxic activities exhibited by A. salicina depended on the chemical composition of the tested extracts.

Discovery of 8-methoxypyrazino[1,2-a]indole as a New Potent Antiproliferative Agent Against Human Leukemia K562 Cells. A Structure-Activity Relationship Study

Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. The indole nucleus, frequently encountered as a molecular fragment in natural products and pharmaceutically active compounds, was employed as the initial building block for the synthesis of a series of pyrazino[1,2-a]indoles 1a-k, variably substituted at the 6, 7, 8 and 9-positions. Compound 1e, bearing the methoxy group at the 8-position of the pyrazino[1,2-a]indole nucleus was identified as a novel potent antiproliferative agent against the human chronic myelogenous leukemia K562 cell line, but it was much less active against several other cancer cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 8- to the 7- or 6-position, to furnish compounds 1f and 1g, respectively, yielded inactive compounds. The analysis of structure-activity relationships observed in the series of investigated compounds may represent the basis for the design of more active molecules.

Relationship correlation of antioxidant and antiproliferative capacity of Cyperus rotundus products towards K562 erythroleukemia cells

A Total Oligomers Flavonoids (TOFs) and ethyl acetate extracts of Cyperus rotundus were analyzed, in vitro, for their antioxidant activity using several biochemical assays: the xanthine (X)/xanthine oxidase (XO), the lipid peroxidation induced by H 2O 2 in K562 human chronic myelogenous leukemia cells and the DNA damage in pKS plasmid DNA assay induced by H 2O 2/UV-photolysis and for their apoptotic effect. TOF and ethyl acetate extracts were found to be efficient in inhibiting xanthine oxidase with IC 50 values of 240 and 185 μg/ml and superoxide anion with IC 50 values of 150 and 215 μg/ml, respectively. Also, all the extracts tested were effective in reducing the production of thiobarbituric acid reactive substances (TBARS) and were able to protect against H 2O 2/UV-photolysis induced DNA damage. The highest activity, measured as equivalents of MDA concentration, was observed in the ethyl acetate extract (MDA = 2.04 nM). In addition, the data suggest that only TOF enriched extract exerts growth inhibition on K562 cells through apoptosis induction. Therefore, these extracts were subjected to further separation by chromatographic methods. Thus, three major compounds (catechin, afzelechin and galloyl quinic acid) were isolated from the TOF enriched extract and five major compounds (luteolin, ferulic acid, quercetin, 3-hydroxy, 4-methoxy-benzoic acid and 6,7-dimethoxycoumarin) from ethyl acetate extract. Their structures were determined by spectroscopic data analysis and comparison with the literature. In addition, we evaluate the biological activities of the catechin, ferulic acid and luteolin. This investigation has revealed that the luteolin was the most active in reducing the production of TBARS (MDA = 1.5 nM), inhibiting significantly the proliferation of K562 cells (IC 50 = 25 μg/ml) and protecting against H 2O 2/UV-photolysis induced DNA damage. In conclusion, the study reveals that the ability of C. rotundus to inhibit the enzyme xanthine oxidase (XO), the lipid peroxidation and to exert apoptotic effect, may explain possible mechanisms by which C. rotundus exhibits its health benefits.

Gambogic acid induces G0/G1 arrest and apoptosis involving inhibition of SRC-3 and inactivation of Akt pathway in K562 leukemia cells

Gambogic acid (GA), a major active component of gamboge, exhibits potent anticancer activity in many kinds of cancer cells. However, the anticancer mechanism of GA is not clearly understood. Here we showed that GA could cause growth inhibition, induce the G0/G1 phase cell cycle arrest and apoptosis in human chronic myelogenous leukemia cell line K562 cells. Since steroid receptor coactivator-3 (SRC-3), overexpressed in many human malignancies including leukemia, is a central target for cancer therapy, we also explored the effects of GA on SRC-3 and SRC-3-regulated gene products in K562. GA treatment downregulated the expression of SRC-3 and then inhibited the activity of Akt kinase and its downstream targets p70 S6 kinase 1 (S6K1) and glycogen synthase kinase 3β (GSK3β) without changes in total protein levels of these three proteins, which thus influenced the expression of the apoptosis related gene Bcl-2 in K562 cells. These results suggest that GA might exhibit its strong antitumor effects via the interruption of SRC-3.

C-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF–IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF–IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

Downregulation of JUNB mRNA expression in advanced phase chronic myelogenous leukemia

JUNB inactivation in transgenic mice results in a myeloproliferative disorder that closely resembles human chronic myelogenous leukemia (CML). It has been reported that downregulation of JUNB expression is a universal phenomenon in patients with CML due aberrant DNA methylation of its promoter. Based on this, we studied methylation and gene expression levels of JUNB in CML. We analyzed the methylation status of the JUNB gene in 6 cell lines and in 102 patients with CML using several bisulfite PCR assays. JUNB expression was analyzed using real-time PCR and gene expression profiling. JUNB methylation was not observed in any of the cell lines studied, and only in 3% of patients with CML. Despite the lack of JUNB methylation, JUNB was expressed at low levels both in CML cell lines (median dCT −6.86; range −5.87 to −9.61), and in patients with CML in blastic phase (BP) (median dCT −3.95; range −1.48 to −6.29) ( p = 0.002). Finally, we evaluated JUNB expression in 82 additional patients with CML by gene expression arrays. We found that JUNB was significantly downregulated in advanced phase CML in contrast to chronic phase CML (median log ratio difference in expression = 0.53). Overall, our results indicate that JUNB expression is downregulated in advanced phase CML through a mechanism independent from DNA methylation.

Synthesis, characterization and in vitro cytotoxicity of the first palladium(II) oxalato complexes involving adenine-based ligands

The first [Pd(L(n))(2)(ox)] xH(2)O oxalato(ox) complexes involving 2-chloro-N6-(benzyl)-9-isopropyladenine (L(1); complex 1), 2-chloro-N6-(4-methoxybenzyl)-9-isopropyladenine (L(2); 2), 2-chloro-N6-(2,3-dimethoxybenzyl)-9-isopropyladenine (L(3); 3), 2-chloro-N6-(2,4-dimethoxybenzyl)-9-isopropyladenine (L(4); 4), and 2-chloro-N6-(4-methylbenzyl)-9-isopropyladenine (L(5); 5) have been synthesized by the reactions of potassium bis(oxalato)palladate(II) dihydrate, [K(2)Pd(ox)(2)].2H(2)O, with the mentioned organic compounds (H(2)ox=oxalic acid; x=0 for 1-3 and 5 or 2 for 4). Elemental analyses (C, H, N), FTIR, Raman and NMR ((1)H, (13)C, (15)N) spectroscopies, conductivity measurements and thermal studies (thermogravimetric and differential thermal analyses, TG/DTA) have been used to characterize the prepared complexes. The molecular structures of [Pd(L(2))(2)(ox)] (2) and [Pd(L(5))(2)(ox)].L(5).Me(2)CO (5.L(5).Me(2)CO) have been determined by a single crystal X-ray analysis. The geometry of these complexes is slightly distorted square-planar with two appropriate L(n) (n=2 or 5) molecules mutually arranged in the head-to-head (2) or head-to-tail (5) orientation. The L(n) ligands are coordinated to the central Pd(II) ion via the N7 atoms. The same conclusions regarding the binding properties of L(1)-L(5) ligands can be made based on multinuclear NMR spectra. In vitro cytotoxicity of the complexes 1-5 has been evaluated against human chronic myelogenous leukaemia (K562) and human breast adenocarcinoma (MCF7) cancer cell lines. Significant cytotoxicity has been determined for the complexes 3 (IC(50)=6.2 microM) and 5 (IC(50)=6.8 microM) on the MCF7 cell line, which is even better than that found for the well-known and widely-used platinum-bearing antineoplastic drugs, i.e. oxaliplatin and cisplatin.

Gamma‐linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant alpha-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.

Molecular imaging of Bcr-Abl phosphokinase in a xenograft model

The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma-imaging using an 111In-labeled anti-phosphotyrosine (APT) antibody, and if the response to treatment with imatinib could be detected using this imaging technique. APT antibody was labeled with 111In using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a nonreceptor tyrosine kinase, xenografts of the human chronic myelogenous leukemia cell line K562 were used. gamma-Scintigraphy of the tumor-bearing mice, before and after imatinib treatment, was obtained 1, 24, and 48 h after they were given 111In-EC-APT (100 microCi/mouse i.v.). 111In-EC-APT is preferentially taken up by Bcr-Abl-bearing tumor cells when compared with 111In-EC-BSA or 111In-EC-IgG1 controls and comparable with the level of uptake of 111In-EC-Bcr-Abl. Imatinib treatment resulted in decreased expression of phospho-Bcr-Abl by Western blot analysis, which correlated with early (4 days after starting imatinib) kinase down-regulation as assessed by imaging using 111In-EC-APT. The optimal time to imaging was 24 and 48 h after injection of 111In-EC-APT. Although tumor regression was insignificant on day 4 after starting imatinib treatment, it was marked by day 14. 111In-EC-APT can assess intracellular phosphokinase activity, and down-regulation of phosphokinase activity predates tumor regression. This technique may therefore be useful in the clinic to detect the presence of phosphokinase activity and for early prediction of response.

Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias

Vorinostat [suberoylanilide hydroxamic acid (SAHA)] is a potent histone deacetylase inhibitor with promising clinical efficacy as an anticancer agent. In this preclinical study, we evaluated combining cytosine arabinoside [1-beta-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias. Cell survival was examined in vitro in HL-60 human myeloid leukemia cells and K562 myeloid blast crisis chronic myelogenous leukemia cells, using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and/or fluorescein diacetate/propidium iodide assays. Drug interactions were analyzed by the combination index method (CalcuSyn) and by a novel statistical method that we developed (SynStat). Cell cycle phase distribution was measured by flow cytometry. Cytotoxic antagonism resulted when vorinostat was combined concomitantly with ara-C; however, when vorinostat was given first followed by a drug-free interval before ara-C treatment, this sequential combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became more pronounced when etoposide was given after vorinostat. Cell cycle analyses revealed that the sequence-dependent interaction of vorinostat and ara-C or etoposide reflected the arrest of cells in G1 or G2 phase during vorinostat treatment and recovery into S phase after removal of vorinostat. These findings using two independent methods to assess drug combination effects provide a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients with advanced or refractory leukemias. CalcuSyn findings were concordant with those of SynStat, validating the use of the latter in analyzing drug interactions.

Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia

Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells--the cells that propagate the leukaemia--whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.